Improved Polysaccharide Production by Homologous Co-overexpression of Phosphoglucomutase and UDP Glucose Pyrophosphorylase Genes in the Mushroom <i>Coprinopsis cinerea</i>

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in <i>Coprinopsis cinerea</i>, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions

Heterologous Expression and Characterization of a Novel Chitinase (ChiEn1) from <i>Coprinopsis cinerea</i> and its Synergism in the Degradation of Chitin

Chitinase ChiEn1 did not hydrolyze insoluble chitin but showed hydrolysis and transglycosylation activities toward chitin-oligosaccharides. Interestingly, the addition of ChiEn1 increased the amount of reducing sugars released from chitin powder by endochitinase ChiIII by 105.0%, and among the released reducing sugars the amount of (GlcNAc)₂ was increased by 149.5%, whereas the amount of GlcNAc was decreased by 10.3%. The percentage of GlcNAc in the products of chitin powder with the combined ChiIII and ChiEn1 was close to that in the products of chitin-oligosaccharides with ChiEn1, rather than that with ChiIII. These results indicate that chitin polymers are first degraded into chitin oligosaccharides by ChiIII and the latter are further degraded to monomers and dimers by ChiEn1, and the synergistic action of ChiEn1 and ChiIII is involved in the efficient degradation of chitin in cell walls during pileus autolysis. The structure modeling explores the molecular base of ChiEn1 action

The Modes of Action of ChiIII, a Chitinase from Mushroom Coprinopsis cinerea, Shift with Changes in the Length of GlcNAc Oligomers

A putative class III endochitinase (ChiIII) was reported previously to be expressed dominantly in fruiting bodies of Coprinopsis cinerea, and its expression levels increased with the maturation of the fruiting bodies. This paper further reports that ChiIII is a novel chitinase with exo- and endoactivities. When the substrate was (GlcNAc)_3–5, ChiIII exhibited exoactivity, releasing GlcNAc processively from the reducing end of (GlcNAc)_3–5; when the substrate was (GlcNAc)_6–7, the activity of ChiIII shifted to an endoacting enzyme, randomly splitting chitin oligosaccharides to various shorter oligosaccharides. This shift in the mode of action of ChiIII may be related to its stronger hydrolytic capacity to degrade chitin in fungal cell walls. The predicted structure of ChiIII shows that it lacks the α+β domain insertion; however, its substrate binding cleft seems to be deeper than that of common endochitinases but shallower and more open than that of common exochitinases, which may be related to its exo- and endohydrolytic activities

The efficiency of GPR3 overexpression.

<p>(A) Immunofluorescence detection of GPR3 overexpression. Control: oocytes were injected with 2.5 mg/ml Myc₆ mRNA solution; Overexpression: oocyets were injected with 2.5 mg/ml Myc₆-GPR3 mRNA solution. Green, Myc₆-GPR3; Blue, chromatin. Bar = 40 µm. (B) Samples from control and overexpression groups were collected to test the expression of Myc₆-GPR3. Control: 150 oocytes injected with 2.5 mg/ml Myc₆ mRNA solution; Overexpression: 150 oocytes injected with 2.5 mg/ml Myc₆- GPR3 mRNA solution. Oocytes were then incubated for 15 h in TCM-199 medium containing 2.5 µM Milrinone before collection or culture for western blotting.</p

The effect of SPC on distribution of GPR3 in porcine oocytes.

<p>Distribution of GPR3 in oocytes after treatment with SPC was revealed by immunofluorescent staining. In the GV stage, GPR3 was mainly distributed at the nuclear membrane and plasma membrane. GPR3 accumulated in the inner cytoplasm and plasma at the pro-MI stage. From MI to MII stages, GPR3 aggregated at the plasma membrane. Green, GPR3; Blue, chromatin. Bar = 40 µm.</p

The effect of GPR3 RNAi on meiotic resumption in porcine oocytes.

<p>(A) Samples from control and RNAi groups were collected to test the efficiency of GPR3-RNAi. Control: 150 oocytes injected with 25 µM control siRNA; RNAi: 150 oocytes injected with 25 µM GPR3 siRNA. Oocytes were then incubated for 24 h or 30 h in the TCM-199 medium with or without 4 mM HX before collection for western blotting. (B) Oocytes cultured with normal medium without HX were injected with 25 µM GPR3 siRNA or control siRNA and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (C) Oocytes treated with 4 mM HX were injected with the GPR3 siRNA or control siRNA and then cultured in the presence of HX for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (D) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected after siRNA injection by western blot using 150 oocytes in each sample. The results are shown along with those of non-injected oocytes cultured without HX. Samples were collected from culture medium with or without HX at 0 h, 24 h and 30 h. (E) cAMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (F) cGMP levels of porcine oocytes in the GPR3 siRNA injection group and control siRNA group after culture with HX at 24 h or 30 h. Dotted line represents cGMP level at time 0. The number “n” on top of the bars indicates the total number of treated oocytes in each group. Data are shown as mean ± SEM of at least three repeated experiments and letters ‘a’ and ‘b’ indicate statistically significant difference (p<0.05).</p

Overexpression of GPR3 inhibits meiotic resumption in porcine oocytes.

<p>(A) Oocytes cultured in normal medium without HX were injected with 2.5 mg/ml Myc₆-GPR3 or control Myc₆ and then cultured for 24 h or 30 h. The GVBD rates of the injected oocytes are shown along with non-injected oocytes. (B) Cyclin B (upper panel) and CDC2 (middle panel) levels were detected by western blot using 150 oocytes in each sample. The results are shown along with those of non-injected oocytes cultured without HX. Samples were collected after culture in medium without HX at 0 h, 24 h and 30 h. (C) cAMP levels of porcine oocytes in the Myc₆-GPR3 injection group and control Myc₆ injection group after culture without HX at 24 h or 30 h. Dotted line represents cAMP level at time 0. (D) cGMP levels of porcine oocytes in the Myc₆-GPR3 injection group and control Myc₆ injection group after culture without HX at 24 h or 30 h. Dotted line represents cGMP level at time 0. The number “n” on top of the bars indicates the total number of treated oocytes in each group. Data are shown as mean ± SEM of at least three repeated experiments and letters ‘a’ and ‘b’ indicate statistically significant difference (p<0.05).</p

Expression and subcellular localization of GPR3 during porcine oocyte meiotic maturation.

<p>(A) Subcellular localization of GPR3 as revealed by immunofluorescent staining. In the GV stage, GPR3 was mainly distributed at the nuclear membrane and plasma membrane. GPR3 accumulated at the inner cytoplasm and plasma membrane at the pro-MI stage. From MI to MII, GPR3 aggregated at the plasma membrane. (B) Expression of GPR3 protein was measured by western blotting. Samples were collected at 0 h, 24 h, 30 h, and 44 h of culture which were the time points when most oocytes had reached the GV, GVBD, MI and MII stages, respectively. Each sample was derived from 150 oocytes. Green, GPR3; Blue, chromatin. Bar = 40 µm.</p